We have successfully crystallized rat liver phenylalanine hydroxylase. We have found that the target size of this enzyme is a monomer in the crude extract, but a dimer in the purified enzyme. The natural cofactor, tetrahydrobiopterin, appears to be capable of inhibiting only the tetrameric form of the enzyme. An enzyme that is involved in the regeneration pathway of tetrahydrobiopterin, 4A-carbinolamine dehydratase, has been further studied by the preparation of an antibody and a subsequent tissue specificity study. This enzyme has recently been suggested by this laboratory, as well as others, to be linked to a new form of hyperphenylalaninemia. 7-tetrahydrobiopterin, a novel isomer of the natural cofactor that is found in relatively high concentrations in certain hyperphenylalaninemic patients, is a partially uncoupled cofactor for phenylalanine hydroxylase. The partially uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase was further studied and shown to follow a similar mechanism to that for the fully uncoupled reaction.